Tissue-based compositions and methods of use thereof

ABSTRACT

Compositions comprising extracellular matrix (ECM) materials and methods of use thereof are disclosed. The compositions may comprise two or more ECM materials derived from different types of tissues, such as, e.g., lung tissue and spleen tissue, formulated for administration to a patient or configured as a medical device for implantation in or application to the patient. The compositions may combine complementary properties of different types of ECM materials for customized patient-specific and/or site-specific tissue repair and/or regeneration.

TECHNICAL FIELD

The present disclosure generally relates to compositions comprisingextracellular matrix (ECM) material and methods of use thereof.

BACKGROUND

Biomaterials have been used in a variety of medical applications asalternative to, or in conjunction with, conventional materials in orderto assist with healing, tissue repair, and other forms of medicaltreatment. Such biomaterials include ECM, a complex structural materialfound within tissues that surround and support cells. The ECM isgenerally made up of three major classes of biomolecules: structuralproteins such as collagen and elastin; other proteins such as laminin,fibronectin, and various growth factors; and proteoglycans. ECM isderived from collageneous tissue and processed for application at thesite of bodily injury. While ECM materials can be used for certainmedical applications, current ECM materials often fail to providesufficient structural integrity and bioactivity.

SUMMARY

The present disclosure includes a composition comprising a firstextracellular matrix material derived from spleen tissue; and a secondextracellular matrix material derived from at least one mammalian tissuechosen from lung tissue, gall bladder tissue, bone marrow tissue,pancreatic tissue, or liver tissue, the second extracellular matrixmaterial being at least partially integrated into the firstextracellular matrix material, wherein at least a portion of thecomposition is in particulate form, and wherein the composition isconfigured for administration to a patient. Embodiments of the presentdisclosure may include one or more of the following features: the firstand/or second extracellular matrix material may be in particulate form;the second extracellular matrix material may be derived from lungtissue; one of the first extracellular matrix material or the secondextracellular matrix material may be in particulate form, and the otherof the first extracellular matrix and the second extracellular matrixmay be in gel form; one of the first extracellular matrix material orthe second extracellular matrix material may be in particulate form, andthe other of the first extracellular matrix material or the secondextracellular matrix material may be in sheet form; the composition maybe configured for application to native tissue of the patient forrepairing the native tissue; the composition may have a rod-like shapeor a tubular shape; at least a portion of the second extracellularmatrix material may be in particulate form, such that the secondextracellular matrix particulate material may be incorporated into thefirst extracellular matrix material, wherein the second extracellularmatrix material may be derived from lung tissue; the composition mayinclude at least two layers, wherein at least one of the layerscomprises the second extracellular matrix particulate materialintegrated into the first extracellular matrix material; the compositionmay further comprise at least one antimicrobial agent such as ionicsilver; the first extracellular matrix material may have a concentrationof at least one growth factor higher than a concentration of the atleast one growth factor of the second extracellular matrix material;and/or the at least one growth factor may be chosen from vascularendothelial growth factor (VEGF), fibroblast growth factor (FGF),platelet-derived growth factor (PDGF), insulin-like growth factor (IGF),or epidermal growth factor (EGF).

The present disclosure further includes a composition comprising a firstextracellular matrix material derived from spleen tissue, and a secondextracellular matrix material derived from at least one mammalian tissuechosen from lung tissue, gall bladder tissue, bone marrow tissue,pancreatic tissue, or liver tissue, the second extracellular matrixmaterial being at least partially integrated into the firstextracellular matrix material, wherein the first extracellular matrixmaterial has a higher growth factor content than the secondextracellular matrix material, and the second extracellular matrixmaterial is at least partially integrated into the first extracellularmatrix material, wherein the first extracellular matrix material is in aform different from a form of the second extracellular matrix material,and wherein the composition is configured for application to nativetissue of a patient for repairing the native tissue. Embodiments of thepresent disclosure may include one or more of the following features:one of the first extracellular matrix material or the secondextracellular matrix material may be in particulate form, and the otherof the first extracellular matrix material and the second extracellularmatrix material may be in gel form; the first extracellular matrixmaterial may have a higher concentration of vascular endothelial growthfactor (VEGF) than the second extracellular matrix material, and thesecond extracellular matrix material may have a higher concentration ofelastin than the first extracellular matrix material; the firstextracellular matrix material may have a higher concentration ofplatelet-derived growth factor (PDGF) than the second extracellularmatrix material, and the second extracellular matrix material may have ahigher concentration of fibroblast growth factor (FGF) than the firstextracellular matrix material; and/or the first extracellular matrixmaterial may have a higher concentration of epidermal growth factor(EGF) than the second extracellular matrix material, and the secondextracellular matrix material may have a higher concentration ofvascular endothelial growth factor (VEGF) than the first extracellularmatrix material.

The present disclosure further includes a composition comprising a firstextracellular matrix material derived from spleen tissue, and a secondextracellular matrix material derived from lung tissue, wherein thecomposition includes at least two layers, at least one of the layerscomprising the second extracellular matrix material at least partiallyintegrated into the first extracellular matrix material, and wherein thecomposition is configured for application to native tissue of a patientfor repairing the native tissue. Embodiments of the present disclosuremay include one or more of the following features: the firstextracellular matrix material may be derived from a reticular portion ofthe spleen tissue, such that the first extracellular matrix material maybe at least partially porous; the first extracellular matrix materialalso may be derived from an outer membrane of the spleen tissue, suchthat the first extracellular matrix material may have a sheet-likeportion and a porous portion, the second extracellular matrix materialbeing integrated into the porous portion of the first extracellularmatrix material; the second extracellular matrix material may be inparticulate form, gel form, or liquid form; the composition may have arod-like shape configured for implantation into the patient; thecomposition may further comprise a third extracellular matrix material,wherein at least one of the layers of the composition may comprise thethird extracellular matrix material in sheet form, wherein the thirdextracellular matrix material may be derived from lung tissue or spleentissue; and/or the composition may have a rod-like shape or a tubularshape.

The present disclosure further includes a composition comprising a firstextracellular matrix material derived from spleen tissue, and a secondextracellular matrix material derived from lung tissue, the secondextracellular matrix material being in particulate form, gel form, orliquid form, wherein the first extracellular matrix material is in aform different from the form of the second extracellular matrixmaterial, and wherein the composition is configured for application tonative tissue of a patient for repairing the native tissue. In someembodiments, at least a portion of the first extracellular matrixmaterial may be in sheet form; and/or the first extracellular matrixmaterial may be derived from a reticular portion of the spleen tissueand an outer membrane of the spleen tissue, such that the firstextracellular matrix material may have a sheet-like portion and a porousportion, wherein the second extracellular matrix material may beintegrated into the porous portion of the first extracellular matrixmaterial.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate various exemplary embodiments andtogether with the description, serve to explain the principles of thedisclosed embodiments. Any features of an embodiment described herein(e.g., composition, medical device, method of treatment, etc.) may becombined with any other embodiment, and are encompassed by the presentdisclosure.

FIG. 1 shows an exemplary composition, in accordance with one or moreembodiments of the present disclosure.

FIG. 2 shows an exemplary composition, in accordance with one or moreembodiments of the present disclosure.

FIGS. 3A-3C show exemplary compositions according to some embodiments ofthe present disclosure, wherein FIGS. 3B and 3C show differentcross-sectional views of a composition as shown in FIG. 3A.

FIGS. 4A and 4B show an exemplary composition according to one or moreembodiments of the present disclosure.

FIGS. 5A and 5B show an exemplary composition according to one or moreembodiments of the present disclosure.

DETAILED DESCRIPTION

The singular forms “a,” “an,” and “the” include plural reference unlessthe context dictates otherwise.

The terms “approximately” and “about” refer to being nearly the same asa referenced number or value. As used herein, the terms “approximately”and “about” generally should be understood to encompass ±10% of aspecified amount or value.

As used herein, the term “therapeutically-effective amount” relates toan amount of a substance (e.g., an agent, compound, material, etc.) thatleads to the desired therapeutic effect(s), and the term“pharmaceutically-effective amount” relates to an amount of a substance(e.g., an agent compound, material, etc.) that leads to the desiredpharmacological effect(s). While individual patient needs may vary,determination of optimal ranges for effective amounts of the substancesdescribed herein (e.g., ECM materials, growth factors, structuralproteins, therapeutic agents, pharmaceutical agents, antimicrobialagents, etc.) is within the skill of the art. For example, suitableamounts or dosages of the substances herein may be selected inaccordance with a variety of factors, including the type, age, weight,sex, diet, medical condition, and/or medical history of the patient.

Wound healing and tissue regeneration is a complex multi-stage processwithin the body. Various and multiple components are involved indifferent stages. Injured or damaged tissue requires multiplecomponents, e.g., structural proteins and signaling factors, to completehealing at the site of injury. Many of these components have beenidentified in ECM materials, including fibrous proteins such asdifferent collagen types, elastin, fibronectin, and laminin. ECM alsomay comprise glycosaminoglycans (GAGs) such as heparin sulfate, andvarious growth factors. During tissue healing/repair/regeneration, thesecomponents play various roles, including, but not limited to,up-regulating and/or down-regulating different stages of the healingprocess. Rather than one single biomolecule, a number of differentcomponents work together to facilitate healing and repair at each stage.Several types of signaling factors are often needed for a specific stageof the healing process to occur. For example, a growth factor may signala stem cell from the host to migrate to the site of injury. The hostcell may need a substrate or structural protein (e.g., a specificcollagen type, laminin, or elastin) for attachment or binding to thesite, and a different growth factor for signaling in order todifferentiate into site-specific tissue.

The present disclosure may address one or more of these challenges,e.g., by combining structural proteins and growth factors or othersignaling or structural molecules from different types and/or sources oftissue in order to facilitate tissue repair at a specific injury site.

Types of Materials

The present disclosure includes compositions comprising ECM materials orother collagen-based materials for promoting tissue repair,augmentation, and/or regeneration. For example, the ECM materials mayserve as a support structure and/or provide signaling factors tofacilitate tissue growth. Materials suitable for the present disclosuremay be derived from any mammalian source tissue comprising ECM or othercollagen-based materials, including, but not limited to, tissues of thespleen, kidney, liver, lung, pancreas, gall bladder, stomach,pericardium, lymph node, bone marrow, dermis, placenta, amniotic sac,dura mater, and any combinations thereof. ECM source material may beused in whole or in part.

The ECM materials may be obtained from tissue by removing the entiretyof the tissue (e.g., an organ) or a portion thereof from the desiredmammalian source, and devitalizing the tissue by removing the cellularcontent of the tissue. The ECM source material may comprise all layersof a type of tissue or an entire organ, for example, or may compriseonly one or more portions of tissue such as the submucosa, the basementmembrane, a tunica layer, the reticular ECM, or the outer membrane ofthe source tissue such as the pleura of the lung or the capsule of thekidney. The ECM materials of the present disclosure may comprise anycombination of these different portions or layers of tissue. Examples oftypes of native tissues suitable for the present disclosure include, butare not limited to, porcine, bovine, ovine, and human tissue. In someembodiments, the composition may comprise one or more non-mammaliantissues such as fish tissue, e.g., fish skin, optionally in combinationwith one or more mammalian tissues.

In some embodiments, the composition may comprise ECM materials derivedfrom two or more different tissue sources and/or two or more differentnative tissues. For example, the tissue sources can be from the samespecies (e.g., ECM materials derived from different types of tissues ofthe same mammal), from different species (e.g., ECM materials derivedfrom the same type of tissue of different types of mammals), or both(e.g., ECM materials derived from different types of tissues ofdifferent types of mammals). In some embodiments, the composition maycomprise spleen ECM and/or lung ECM from the same species or differentspecies. For example, embodiments of the present disclosure may include,but are not limited to, compositions comprising the following:

-   -   Porcine spleen ECM and porcine lung ECM    -   Bovine spleen ECM and porcine spleen ECM    -   Bovine lung ECM and porcine spleen ECM    -   Bovine lung ECM, bovine spleen ECM, and porcine spleen ECM        The composition may comprise spleen ECM and at least one other        ECM material chosen from lung, gall bladder, bone marrow,        pancreas, or liver. For example, the composition may comprise        spleen ECM, lung ECM, and/or at least one other ECM material.        Other combinations of ECM materials will be apparent in view of        the disclosure herein.

In addition to ECM materials, the compositions may comprise one or moreother compounds or materials, such as resorbable synthetic or naturalmaterials, non-resorbable synthetic or natural materials, polymers,metals, bone material, allograft tissues or bones, antimicrobial agents,therapeutic agents, pharmaceutical agents (drugs), or any combinationthereof. In some embodiments, the composition may be formulated foradministration to patient and/or configured as a medical device orcomponent thereof for application or implantation into a patient.

In some embodiments, for example, the composition may comprise spleenand lung ECM, optionally in combination with one or more other types ofECM materials and/or one or more non-ECM components. For example, acomposition of spleen and lung ECM may comprise one or more other typesof ECM materials including, but not limited to, heart membrane ECM,pericardium ECM, pancreas ECM, fascia ECM, dura mater ECM, omentum ECM,gall bladder ECM, amniotic sac ECM, kidney capsule ECM, liver ECM, orbone marrow ECM. In at least one embodiment, the composition maycomprise spleen ECM, lung ECM, and one or more non-ECM components chosenfrom polymers, hydrogels, or hyaluronic acid.

Preparation/Treatment of ECM Materials

Methods of preparing devitalized or acellular tissue may includephysical, chemical, and/or biological processes. For example, mammaliantissue may be harvested and subjected to a physical cleaning process toremove fat, muscle, and other cellular material extraneous to the ECM.Such physical processes may include machine-based mechanical actionsand/or force applied manually. In addition or alternatively, the tissuemay be subjected to chemical and/or biological processes to rupturecells and remove cellular material. For example, the tissue may beexposed to one or more chemical or biological agents including, but notlimited to, an acid (e.g., HCl, acetic acid, peracetic acid), a base(e.g., NaOH), a chelating agent (e.g., ethylenediaminetetraacetic acid(EDTA), ethylene glycol tetraacetic acid (EGTA)), a detergent (e.g.,sodium dodecyl sulfate, Triton-X, CHAPS), and/or an enzyme (e.g.,nuclease, trypsin, collagenase). Further examples of decellularizationprocesses include high pressure homogenization, and hypotonic orhypertonic washes for rupturing cells in ECM materials and washing awaythe cellular material. The ECM materials may be dried, e.g., at ambienttemperature and pressure or in vacuum, frozen, and/or stored prior touse. Methods of drying ECM materials suitable for the present disclosureinclude desiccation chambers, controlled humidity chambers, forced airflow drying, and lyophilization (freeze-drying).

The process(es) involved in decellularization may be selected topreserve one or more biological materials of interest. Decellularizationmay be different for each type of tissue, based on the desiredcomponents and/or structure to be retained, and the treatment suitablefor retaining those components and/or structure. Embodiments of thepresent disclosure may comprise ECM materials derived from at least twodifferent types of tissue, each tissue subjected to one or moredecellularization processes selected to retain particularcharacteristics of the tissue.

In some embodiments, for example, lung tissue may be decellularized withdetergents such as sodium dodecyl sulfate or Triton-X to retain as muchelastin from the material as possible while sacrificing some of thegrowth factor content. In addition to elasticity, other benefits tousing lung ECM may include liquid impermeability, e.g., to prevent fluidwithin the composition from leaking out and/or to prevent bodily fluidsfrom permeating into or through the composition. In addition, lung ECMmay be processed in such a way to provide one relatively smooth surfacethat discourages the attachment of patient tissue to the composition,while the opposite surface may have a more porous structure thatfacilitates cell attachment. Lung ECM also may be particularly suitablefor applications that require materials with stretch and/or reboundproperties, such as bladder augmentation, vascular graft, or vascularpatch applications. Further, lung ECM may be relatively thin (e.g.,derived from lung tissue that is relatively thin in comparison to othertypes of tissues), which may provide for a beneficial strength-to-weightratio in comparison to other types of ECM materials.

Spleen tissue, on the other hand, may be decellularized to preserve asmuch growth factor content as possible by treating the tissue with amore gentle acid wash, such as peracetic acid having a concentrationranging from about 0.01% to about 5.00%, such as about 0.1%. Thedecellularized spleen tissue may be rinsed with buffered saline and/orwater to retain a maximum amount of growth factor. The reticularstructure of spleen ECM may provide interstitial spaces suitable foraccommodating other types of ECM materials (e.g., in particulate, gel,or paste form), biomolecules, antimicrobial agents (see discussionbelow), and/or pharmaceutical agents. Further, the reticular structureof spleen ECM, e.g., in combination with the growth factors retained inthe matrix, may foster and support the growth of new cells within thematrix to promote tissue grafting. For example, spleen ECM may beparticularly suitable for hemostasis and reconstructive surgeryapplications.

In some embodiments, spleen tissue may be processed to generate ECMmaterial comprising only a portion, e.g., a fibrous portion, of thenative tissue. For example, only the outer membrane of the spleen may beused to generate spleen ECM in sheet form. Further, for example, onlythe reticular ECM of the spleen may be used to generate an openstructure fibrous ECM sheet. In yet another example, both the outermembrane and the reticular ECM component of the spleen may be used suchthat they remain intact, e.g., to generate an ECM material having arelatively more dense, sheet-like structure on one side with arelatively more porous or fibrous sponge-like structure on the otherside.

During processing, ECM materials may shrink when dried and then expandto some degree when they are rehydrated, e.g., as they absorb fluid. Forexample, an ECM material in sheet form may be about 200 μm thick whendry, and may increase to a thickness from about 300 μm to about 500 μmwhen rehydrated. The natural fibrous architecture of tissues such as thespleen may allow for more expansion, e.g., since the naturally-occurringreticular ECM component generally has “dead space” to allow forcompression and expansion when rehydrated. ECM materials may beprocessed to adjust for compression/rehydration/expansioncharacteristics of the native tissue.

In some embodiments, the composition may comprise a plurality of ECMmaterials, wherein at least one of the ECM materials comprises adifferent variety of components and/or a different amount orconcentration of a given component than another ECM material in thecomposition. Further, in some embodiments, the composition may comprisetwo or more ECM materials that comprise some or all of the same varietyof components, but different amounts or concentrations of thosecomponents. In some embodiments, the composition may comprise two ormore ECM materials that comprise some or all of the same variety ofcomponents and/or substantially the same amount or concentration (or asimilar amount or concentration) of a given component as another ECMmaterial in the composition. These components may include proteins,glycoproteins, glycosaminoglycans, proteoglycans, cytokines, and/orgrowth factors. For example, the composition may comprise ECM materialshaving substantially the same, similar, or different amounts of one ormore of the following components: collagen, elastin, fibronectin,laminin, heparin sulfate, fibroblast growth factor (FGF), vascularendothelial growth factor (VEGF), transforming growth factors alpha(TGF-α), transforming growth factors beta (TGF-β), platelet-derivedgrowth factor (PDGF), epidermal growth factor (EGF), keratinocyte growthfactor (KGF), bone morphogenetic proteins (BMPs), epidermal growthfactor (EGF), brain-derived neurotrophic factor (BDNF), growthdifferentiation factor-9 (GDF9), hepatocyte growth factor (HGF),insulin-like growth factor (IGF), nerve growth factor (NGF), skeletalgrowth factor (SU), osteoblast-derived growth factor (BDGF), cytokinegrowth factors (CGF), stem cell derived factor (SDF), stem cell factor(SCF), placental growth factor (PGF), and/or interleukins of any typeand within any family (e.g., IL-1, IL-2, etc.).

Biological components may be detected and quantified in the ECMmaterials and/or in the native tissues via enzyme-linked immunosorbentassay (ELISA), or any other suitable technique. Table 1 lists growthfactors measured in different porcine ECM materials: small intestinesubmucosa (SIS), urinary bladder submucosa (UBS), stomach submucosa(SS), spleen, and lung. The SS, spleen, and lung measurements aredescribed in Example 1.

TABLE 1 Growth factors in porcine ECM materials. SIS (ng/g)¹ UBS (pg/g)²SS (ng/mg)^(3,4) Spleen (ng/mg)^(3,5) Lung (ng/mg)^(3,6) VEGF 0.77 104.6± 25.4  10.5 ± 1.6  8.7 ± 1.2 11.4 ± 2.7  FGF 97.9 ± 11.7 4.0427 ±0.0075 0.6 1.0 1.1 TGF-β 768.1 ± 182.1 8.8490 ± 0.001  — — — PDGF —285.62 ± 4.54  6.1 6.5 6.1 IGF — 6.9 ± 3.4 0.4 1.6 2.8 EGF — 0.99 ± 0.153.5 ± 2.4 5.2 ± 0.6 5.0 ± 1.6 KGF — 158.8 ± 45.4  — — — ¹Hodde et al.,Endothelium, vol. 8, pp. 11-24 (2001); McDevitt et al., J. Biomed.Mater. Res., vol. 67A, pp. 637-640 (2003); Hodde et al., Wounds, vol.13, pp. 195-201 (2001). ²Chun et al., Biomaterials, vol. 28, pp.4251-4256 (2007). ³Highest value measured of any extraction liquid.⁴Values (ng/mg) with PBS extraction, VEGF = 8.7 ± 0.4, PDGF = 6.1, EGF =3.5 ± 2.4; with RIPA buffer extraction, VEGF = 6.3 ± 0.2, IGF = 0.1, EGF= 1.9 ± 0.7; with acetic acid extraction, VEGF = 10.5 ± 1.6, FGF = 0.6,PDGF = 3.4, IGF = 0.4, EGF = 2.2 ± 0.8. ⁵Values (ng/mg) with PBSextraction: VEGF = 8.2 ± 0.1, PDGF = 6.5, IGF = 0.1, EGF = 5.2 ± 0.6;with RIPA buffer extraction: VEGF = 6.6 ± 0.4, IGF = 0.2, EGF = 5.0 ±0.6; with acetic acid extraction: VEGF = 8.7 ± 1.2, FGF = 1.0, PDGF =6.4, IGF = 1.6, EGF = 4.1 ± 0.5. ⁶Values (ng/mg) with PBS extraction:VEGF = 3.8 ± 0.2, PDGF = 6.1, IGF = 0.03, EGF = 5.1 ± 1.6; with RIPAbuffer extraction: VEGF = 3.2 ± 0.1, IGF = 0.1, EGF = 3.6 ± 0.8; withacetic acid extraction: VEGF = 11.4 ± 2.7, FGF = 1.1, PDGF = 4.9, IGF =2.8, EGF = 3.5 ± 1.0.

The growth factor content measured may vary according to the type ofextraction liquid and/or method used. For example, in some embodiments,the content of different growth factors in stomach submucosa ECM mayrange from about 6.3±0.2 ng/mg to about 10.5±1.6 ng/mg of VEGF, fromabout 3.4 ng/mg to about 6.1 ng/mg of PDGF, from about 0.1 ng/mg toabout 0.4 ng/mg of IGF, and/or from about 1.9±0.7 ng/mg to about 3.5±2.4ng/mg of EGF. In some embodiments, the content of different growthfactors in spleen ECM may range from about 6.6±0.4 ng/mg to about8.7±1.2 ng/mg of VEGF, from about 6.4 ng/mg to about 6.5 ng/mg of PDGF,from about 0.1 ng/mg to about 1.6 ng/mg of IGF, and/or from about4.1±0.5 ng/mg to about 5.2±0.6 ng/mg of EGF. In some embodiments, thecontent of different growth factors in lung ECM may range from about3.2±0.1 ng/mg to about 11.4±2.7 ng/mg of VEGF, from about 4.9 ng/mg toabout 6.1 ng/mg of PDGF, from about 0.03 ng/mg to about 2.8 ng/mg ofIGF, and/or from about 3.5±1.0 ng/mg to about 5.1±1.6 ng/mg of EGF.

Spleen and lung ECM each generally have greater growth factor contentthan SIS, UBS (or urinary bladder matrix, UBM), and SS ECM. While eachof these five types of ECM materials comprises multiple collagen types,lung ECM generally has the greatest quantity of elastin. Elastin mayprovide better clinical results when used in applications requiringstretch and rebound, such as compliance in a vascular graft or vascularpatch application. SIS, UBS/UBM, SS, and lung pleura ECM all naturallyoccur in sheet form, whereas the spleen can be processed to retain muchof the reticular ECM component. The reticular structure provides for anatural three-dimensional ECM with a macroscopic fibrous ECM network.This natural macro-fibrous spleen reticular ECM may be beneficial for avariety of medical applications, such as repair of defects in plasticand reconstructive surgery, use as a layer in a surgical graft such ashernia repair to facilitate better tissue ingrowth, as a hemostasisdevice, or as a natural scaffold for better incorporation of other ECMor non-ECM components, such as particulates, gels, and polymers.

Compositions according to the present disclosure may comprise the sameor different amounts of each ECM material. In some embodiments, thecomposition may comprise two different ECM materials, e.g., in a ratioranging from about 50:50 (i.e., 1:1) to about 5:95 (i.e., 1:19). Forexample, the composition may comprise a 50:50 ratio of two different ECMmaterials (e.g., spleen ECM and lung ECM), or a ratio of 60:40, 70:30,80:20, 90:10, 40:60, 30:70, 20:80, 10:90, or any other ratios inbetween. For example, the composition may comprise lung and spleentissues in a 1:3 ratio (e.g., about 25% lung ECM and about 75% spleenECM, or about 25% spleen ECM and about 75% lung ECM). In someembodiments, the composition may comprise more than two different ECMmaterials, e.g., three, four, five or more ECM materials, in equal orunequal amounts. For example, the composition may comprise threedifferent ECM materials having a ratio of 40:40:20, 30:30:40, or20:20:60, among other possible ratios. In some embodiments, for example,the composition may comprise about 25% spleen ECM, about 25% lung ECM,and about 50% gall bladder ECM. Any desired ratio may be selected basedon the desired final composition.

The ratios of ECM materials in the composition may be chosen based onthe individual characteristics of the ECM materials and desiredapplication for the composition. Regenerative medicine applications forwhich the ratios of ECM materials can be adjusted, e.g., to takeadvantage of the physical, mechanical, and/or biological characteristicsof each ECM material include, but are not limited to, wound dressings,dura repair, fistula plugs, myocardial patches, myocardial injections,heart valve repair, tympanoplasty grafts, nasal septal defect repair,hernia or body wall repair, hemostasis grafts, urology slings, trachealgrafts, esophageal grafts, lung patches, small bowel grafts, staplebolsters, nerve grafts, spinal cord repair, nerve cuff, nerve guide,pelvic floor grafts, amniotic sac patches, cornea repair, cartilagerepair, bone repair, tendon/ligament repair, muscle repair, plastic andreconstructive surgery applications, lip augmentation, facialaugmentation, nipple reconstruction, bile duct repair, ureter repair,urethra repair, and vascular access graft. By varying the ratio andtypes of ECM materials applied in a medical application, higher levelsof signaling factors and/or other components needed for each specifictissue repair type may be provided.

In wound care applications, for example, a higher ratio of spleen ECM tolung ECM may be desired to utilize a relatively higher EGF content inspleen tissue while also taking advantage of a relatively higher VEGFcontent in lung tissue. For vascular graft applications, a higher ratioof lung ECM to spleen ECM may be desired to take advantage of higherelastin content and lower gas/liquid permeability of lung tissue, whileutilizing a relatively higher PDGF content in spleen tissue. In someembodiments, the composition may comprise a tissue graft compositioncomprising a plurality of ECM materials, wherein at least two of the ECMmaterials are derived from different tissue sources and have differentgrowth factor content or protein content.

Forms of Materials

The compositions may comprise ECM materials or collagen-based materialsin a variety of different forms, and may be administered to a patient asa formulation (e.g., a liquid for injection or powder for inhalation),or delivered as part of a medical device (e.g., a topical bandage orimplantable medical device). For example, the ECM materials orcollagen-based materials may be formed into a sheet, rod, tube,three-dimensional construct, mesh, sponge, liquid, gel, hydrogel,emulsion, particles, powder (e.g., fine particles) suspension, paste,putty, dispersion, foam, or any combination thereof, among otherpossible forms. The composition may comprise a combination of ECMmaterials or collagen-based materials in different forms, such as sheetand powder, powder and gel, sheet and gel, sponge and liquid, sponge andgel, sheet/powder/gel, sheet/powder/sheet, sheet/gel/sheet,sheet/gel/powder/sheet, sponge/sheet, sponge/gel, sponge/powder,sponge/powder/sheet, sponge/gel/sheet, sponge/powder/gel/sheet,foam/powder, foam/gel, foam/sheet, foam/powder/sheet, andfoam/powder/gel/sheet, among other combinations.

In some embodiments, the composition may comprise a multilayer ECMmaterial, e.g., two or more layers of ECM materials coupled together,wherein each layer may comprise ECM materials in the same or differentforms, and/or from the same or different sources. The layers may bebonded together or crosslinked, such as crosslinking multiple sheetlayers together via a chemical process. Crosslinking can be achievedthrough various methods and processes generally known to one of ordinaryskill in the art, including chemical and/or biochemical processes,temperature-based methods, pressure-based methods, processes thatinclude exposure to light, and energy-based methods. Crosslinking may beachieved via differential crosslinking, e.g., by performing targetedcrosslinking to achieve bonding of different tissue layers or ECMcomponents at specific locations or regions. Crosslinking may be used togenerate different elasticity properties and/or other mechanicalproperties at the crosslinked sites or regions, this allowing thecomposition to be tailored for a specific application, or to generate adesired size and/or shape for a specific application.

In some embodiments, the ECM materials may retain the structure of thenative tissue. For example, a sheet may be produced by decellularizingnative tissue that has a sheet-like structure, such as membrane layers(e.g., fascia or dermis) or submucosa, while a sponge or scaffold-likestructure may be produced by decellularizing native tissue that hasinterstitial structure, e.g., organs such as the spleen, kidney,pancreas, or liver. Further, for example, rod-like ECM materials may beproduced from native tissues having a generally rod-like or cylindricalstructure such as tendons and nerves; and tubular ECM materials may beproduced from native tissues have a tubular structure, such as bloodvessels, the trachea, or the esophagus.

In some embodiments, the ECM materials or collagen-based materials maybe manipulated or chemically altered into a form substantially differentfrom the form of the native tissue. For example, the composition maycomprise one or more ECM materials or collagen-based reduced toparticulate form via grinding, crushing, milling, or other mechanicalprocess. The particles may be used in the composition directly, or maybe combined with a suitable liquid or gel to form a paste or dispersionfor administration. Further, for example, one or more ECM materials maybe dissolved into a liquid or gel, e.g., via enzymatic digestion orother biological or chemical process. The liquid or gel may be used inthe composition directly, or may be combined with a suitable liquid orgel to form an emulsion for administration. In some embodiments, thestructure of the ECM material(s) may be modified, e.g., via a chemicalprocess to denature proteins of the ECM, to provide for increasedporosity.

For particulate compositions (also referred to as powders herein), theparticles may range from about 100 nm to about 2000 μm in diameter. Theparticle size distribution of a composition may be selected based on thedesired application. For example, particles ranging from about 100 nm toabout 5 μm in diameter, e.g., from about 1 μm to about 5 μm in diameter,may be suitable for administration via inhalation, while particlesranging from about 1000 μm to about 2000 μm in diameter may be suitablefor use in filling voids or augmentation applications. Further, forexample, particles ranging from about 50 μm to about 100 μm in diametermay be suitable for a variety of therapeutic injection applications,while particles ranging from about 100 μm to about 1000 μm in diametermay be suitable for topical wound applications, and particles rangingfrom about 10 μm to about 50 μm may be suitable for corneal repairapplications (e.g., the particles being combined with a suitable liquidand delivered to the eye via a suspension, such as with eye drops).These size ranges are intended as general guidelines only, and may varyaccording to the medical application and/or particular needs of apatient.

In some embodiments, combinations of ECM materials may be manipulatedinto single composite form or construct. For example, pieces or stripsof different ECM sheet materials coupled together to form a compositeECM material sheet. The pieces or strips of different ECM materials maybe stitched, stapled, or coupled together with a suitable adhesive, orchemically cross-linked or bonded together. In some embodiments, thecomposition may comprise portions of sheet-like spleen ECM coupled toportions of sheet-like lung ECM, e.g., to form a composite ECM sheet.Similarly, portions of different ECM materials may be coupled togetherto form a single rod, tube, three-dimensional construct, mesh, sponge,or other suitable form of ECM material.

As mentioned above, the structures and components of different ECMmaterials may provide different benefits, such that combining two ormore types of ECM materials may provide compositions uniquely tailoredto the specific needs of a patient. The compositions may be designed totake advantage of the signaling components naturally occurring in thetissues from which the ECM materials are derived, including, but notlimited to, VEGF, FGF, EGF, TGF-β, PDGF, and/or IGF.

For example, the composition may comprise two or more ECM materials inparticulate form, with the ratios of the ECM materials adjusted toprovide a composition with a desired content of desired components,including, but not limited to, growth factor and/or other signalingcomponents. The composition may be applied in particulate form to a sitein need of repair, or may be injected/applied as a suspension orparticulates in combination with a carrier (e.g., liquid solution).Applications for particulate compositions include, but are not limitedto, nerve repair, spinal disc repair, arthritis treatment, hairrestoration, bone regeneration, bone augmentation, cartilage repair,tendon repair, ligament repair, burn treatment, myelin sheathregeneration, cornea repair, and lung repair.

Antimicrobial Agent(s)

In some embodiments, the composition may have antimicrobial properties,e.g., to reduce, eliminate, prevent, or otherwise control microbialactivity upon application of the composition to a patient. For example,the composition may comprise one or more antimicrobial agents. Suitableantimicrobial agents include, but are not limited to, silver andcompounds and alloys thereof (e.g., silver, silver oxide, silvernitrate, silver sulfazidine, silver-imidazolate, silver phosphate), zincand compounds and alloys thereof (e.g., zinc, zinc chloride, zinc oxide,zinc sulfate monohydrate, zinc-hydroxyapatite, brass), copper andcompounds and alloys thereof (e.g., copper, brass, bronze), bismuth andcompounds and alloys thereof, biguanide compounds (e.g.,polyhexamethylene biguanide, chlorhexadine, polyaminopropyl biguanide,alexidine), berizalkonium chloride, triclosan(5-chloro-2-(2,4-dichlorophenoxy)phenol), antibiotic drugs such asneomycin and bacitracin, honey, coconut, coconut-based products,essential oils, and plant extracts. In some embodiments, a combinationof agents may be used, e.g., to provide a wide spectrum of antimicrobialactivity. For example, the antimicrobial agent(s) may be effectiveagainst bacteria, yeast, fungi, and/or extracellular viruses, e.g., byinhibiting microbial cell activities, transport and/or reproduction. Theantimicrobial agent(s) may be selected based on compatibility with othercomponents of the composition and/or the medical needs of the patient.In some embodiments, the amount of antimicrobial agent(s) in thecomposition may be chosen based on the desired antimicrobial effect onthe patient to be treated with the composition.

Antimicrobial agents may be applied to any surface of, or otherwiseincorporated into, one or more ECM materials of the composition or thecomposition as a whole. While the following discussion will refer todeposition of antimicrobial agents on ECM materials generally, it isunderstood that the antimicrobial agents may be applied alternatively oradditionally to multiple types of ECM materials simultaneously, such asapplication to a composition comprising different types of ECMmaterials.

The antimicrobial agent may comprise a solid, a liquid, a solution(e.g., a solid dissolved or dispersed in a liquid solvent), or a vapor.To deposit antimicrobial agents in liquid form, or a solid in solution,the ECM materials may be immersed in the liquid/solution or theliquid/solution applied to various portions of the ECM materials. TheECM materials may be dried, e.g., via evaporation, upon heating, underreduced pressure, in controlled humidity conditions or desiccationchambers, or under forced airflow. In the case of a solution, forexample, evaporating the solvent may leave behind the solidantimicrobial agent as a deposit or thin layer on the ECM materials. Insome embodiments, the antimicrobial-coated ECM materials may be brokendown into smaller pieces or particles to be incorporated into acomposition and/or for administration to a patient in particulate form.The antimicrobial agent may be chemically and/or biochemically bound tothe ECM substrate.

The method of applying the antimicrobial agent(s) may be selected basedon the form of the ECM materials or composition. In the case of a sheet,for example, an antimicrobial agent may be applied to either or bothsides of the sheet. For a multilayer structure (see, e.g., FIGS. 1 and3, discussed below), the antimicrobial agent may be applied solely tothe outermost layers, a combination of outer and inner layers, or solelythe inner layers. For example, a multilayer composition may be assembledby treating one or more sheets with an antimicrobial agent, layering thesheets to form multiple layers, and optionally crosslinking the layerstogether. In the case of a reticular or scaffold-like structure, the ECMmatrix may be immersed in a solution comprising the antimicrobial agent,such that the solution may permeate the matrix, or the antimicrobialagent in particulate form may be directly incorporated into the matrix.In some embodiments, an antimicrobial agent may be localized within theECM matrix by applying the solution to select portions of the matrix.

In some embodiments, the antimicrobial agent may be deposited from thevapor phase. For example, the antimicrobial material to be deposited maycomprise crystals having a crystalline lattice structure generated inthe vapor phase, e.g., via evaporation or sputtering, and transportedinto a volume in which the temperature is controlled. Atoms of theantimicrobial material may collide with the working gas, causing them tolose energy, such that the antimicrobial material is condensed from thevapor phase onto a cooled substrate, such as a liquid nitrogen-cooledfinger. For silver, deposition may be conducted at low substratetemperatures, e.g., ranging from about −10° C. to about 100° C.

In some embodiments, the composition may comprise silver particles(microparticles and/or nanoparticles). The silver may be in ionic form,e.g. particles comprising a silver salt. The silver may be applied as aspray coating or added as a solution to the ECM materials and thendried. For example, the silver particles may be dispersed in water orother solvent to form a solution, the solution applied to one or moresurfaces of the ECM materials, and the solvent allowed to evaporate suchthat the silver particles remain as an antimicrobial coating on the ECMmaterials. Further, for example, the particles may be embedded directlywithin an ECM matrix without dispersing the particles in solution. Insome embodiments, the composition may comprise silver-coated ECMparticles, e.g., produced by coating ECM particles or reducingcoated-ECM materials to particulate form. In some embodiments, silvercan be applied by other coating methods such as ion beam deposition. Thesilver can be incorporated before or after the ECM materials are dried(e.g., following a decellularization process), or may be incorporatedinto a particulate, liquid, emulsion, or gel form of the ECM materials.

In some embodiments, the silver content of the ECM materials (surfaceconcentration) may range from about 1 mg/100 cm² to about 1000 mg/100cm², such as from about 1 mg/100 cm² to about 100 mg/100 cm². In someembodiments, the silver content of the ECM materials (massconcentration) may range from about 1 mg/g to 100 mg/g, such as fromabout 1 mg/g to about 20 mg/g. In some embodiments, the silver mayprovide a coating on the ECM materials, wherein the thickness of thesilver coating may range from about 100 Å to about 5 μm, such as fromabout 500 Å to about 2 μm.

Exemplary Administration/Applications

Compositions according to the present disclosure may be designed forexternal and/or internal administration to a patient. The compositionsmay be applied to an anatomical site other than that of the ECM sourcematerial(s), or to the same anatomical site(s). The patient tissue inneed of repair, augmentation, or regeneration may include, but is notlimited to, skin, muscle, tendon, ligament, nerve, vascular, dura mater,cornea, bone, heart, liver, lung, spleen, pancreas, stomach, intestine,or brain tissue. The ECM materials may be derived from tissue that isallogeneic, autologous, or xenogeneic to the patient being treated.

In some embodiments, at least a portion or the entire composition may bebiodegradable/resorbable, e.g., such that the composition need not beremoved after application or implantation into a patient. For example,at least a portion of the composition may be configured to be absorbedor resorbed, to dissolve, or to degrade within about 24 hours, 48 hours,72 hours, 1 week, 2 weeks, 1 month, 3 months, or 6 months of applicationor implantation. The ECM materials may be entirely resorbable, onlypartially resorbable, or non-resorbable. For example, the ECM materialsmay be processed to modify their absorption characteristics, such as viacrosslinking with glutaraldehyde or other suitable crosslinking agent.In addition to ECM materials, which may be resorbable or non-resorbable,the composition may comprise one or more natural or syntheticresorbable/biodegradable materials, such as collagen, hydroxylapatite,polylactides, polyglycolides, polycaprolactones, hyaluranic acid,gelatin, bioactive glass, tricalcium phosphate, soft tissue allografts,or hard tissue allografts.

The composition may be configured for temporary application, e.g., atemporary bandage or implantable medical device, to be removed after acertain amount of time, such as after about 1 week, 2 weeks, 1 month, 3months, 6 months, 9 months, or 1 year. For example, the composition maycomprise a hemostasis bandage to help stop bleeding of a wound, suchthat the bandage may be removed once bleeding has stopped and/or forsurgical repair of the wound. Further, for example, the composition maycomprise a coating on a medical device configured for temporaryimplantation in the body, such as a stent or a catheter, or a pacemakerlead intended to be removed at a later date. The ECM materials of thecoating may help to prevent infection (e.g., in conjunction with anantimicrobial agent of the composition), and/or may facilitate contactbetween the medical device and the native tissue of the patient.

In some embodiments, the composition may be configured for permanentimplantation, not to be removed from the patient, and/or may comprise acoating of a medical device configured for permanent implantation.Similar to the above, coatings used for permanent implantation of amedical device may help to prevent infection and/or facilitate contactwith native tissue of the patient.

Embodiments of the present disclosure include identifying a site ofdefect or wound, e.g., in mammalian tissue, such as in a patient,providing a composition comprising two or more ECM materials (e.g.,spleen ECM in combination with one or more of lung ECM, gall bladderECM, bone marrow ECM, pancreas ECM, or liver ECM), contacting the sitewith a therapeutically-effective amount of the composition, and healingor regenerating tissue at the site. In some embodiments, the compositionmay comprise an antimicrobial agent such as ionic silver.

Compositions according to the present disclosure may include one or moretherapeutic agents and/or one or more pharmaceutical agents. The typesand amounts of therapeutic agents and/or pharmaceutical agents may bechosen based on the desired therapeutic or pharmacological effect(s) ona patient to be treated with the composition. Exemplary therapeuticagents may include, but are not limited to, moisturizing agents, dryingagents, and soothing agents. Exemplary pharmaceutical agents include,but are not limited to, steroids, hormones, analgesics,anti-inflammatory agents, and chemotherapy drugs. Pharmaceutical andtherapeutic agents may be chosen based on the intended application ofthe composition. For example, the composition may comprise apharmaceutical agent to help prevent narrowing (stenosis) of bloodvessels for vascular applications, or to help prevent cell adhesion,e.g., by serving as an adhesion barrier for hernia repair or otherinternal body cavity repair applications. The compositions may be usedfor drug delivery, to deliver one or more pharmaceutical agents to aspecific anatomical site or area. For example, the compositions may betailored to dissolve, degrade, or be absorbed by the body at a rate thatallows for elution of one or more pharmaceutical agents at a controlledor desired rate, e.g., to deliver a pharmaceutically-effective amount ofthe pharmaceutical agent(s). In some embodiments, the composition maycomprise small “pellets” or dense cylinders of ECM dosed with one ormore chemotherapy drug(s). The composition may be formulated forinjection or to be surgically implanted at a tumor site, such that thecomposition releases the drug(s) at the desired site, while limiting orminimizing trauma due to exposure to other tissues and/orbody-system-wide side effects.

The following describes various exemplary compositions, includingdifferent forms and combinations of ECM materials, and methods of usethereof. While some of the examples describe combinations of specifictypes of ECM materials (e.g., spleen and lung ECM), other combinationsof ECM materials are likewise possible and encompassed herein.

The composition may comprise one or more ECM materials in sheet form,including sheets with particular texture or structure to promote tissuegrowth, such as a mesh-like or scaffold-like structure. For example, thecomposition may include a scaffold-like structure for promotingrestoration of tissue when implanted at anatomical site in a patient.The composition may have a multilayer configuration (e.g., two or moresheet layers bonded, crosslinked, or otherwise coupled together), and/ora sandwich configuration (e.g., two or more sheets bordering orenclosing a material therebetween, or a single sheet folded over onitself to encase a material). The composition may be designed forexternal application, internal application, or both. In someembodiments, the sheets may be treated with an antimicrobial agent asdiscussed above to provide the composition with antimicrobialproperties.

FIGS. 1 and 2 illustrate exemplary compositions comprising ECM sheetmaterials in accordance with the present disclosure. FIG. 1 shows amultilayer composition 10 comprising layers that have different types oftissue structure. As shown, the composition may comprise a first layer12 of ECM material having a sheet-like structure, and a second layer 14of ECM material having both sheet-like and reticular, fibrousmatrix-like structure. In some embodiments, the second layer 14 maycomprise only matrix-like structure. The two layers may be derived fromthe same type of tissue (e.g., a sheet-like layer and matrix layer ofspleen ECM coupled together) or different types of tissues (e.g., asheet-like layer of lung ECM coupled to a reticular layer of spleenECM). The composition may comprise more than two layers, e.g.,alternating sheet-matrix layers, two or more sheet-like layers, two ormore matrix layers, etc.

In some embodiments, for example, the composition may comprise a tissuegraft or wound dressing (e.g., a bandage, patch, pad, compress, medicaltape, etc.) that includes an outer sheet layer of lung ECM and innermatrix layer of spleen ECM, thus combining outer liquid impermeabilityfrom the lung ECM with an inner intricate network from the spleen ECMcapable of accommodating materials to promote cell regeneration.

FIG. 2 shows an exemplary composition 20 comprising ECM materials havingboth sheet-like structure and matrix structure. For example, the ECMmaterials may be derived from spleen tissue, such that during thedecellularization process the fibrous network or matrix structure 24remains intact and attached to the membrane layer 22 on either side ofthe reticular matrix 24. The composition 20 may comprise a second typeof ECM material 25 incorporated into the interstitial spaces within thereticular matrix structure 24. For example, the composition 20 maycomprise particles of lung ECM and/or bone marrow ECM embedded withinthe reticular spleen matrix, a combination of lung ECM and spleen ECMparticles, a combination of lung ECM in particulate form and in gelform, or a combination of lung ECM in particulate form and spleen ECM ingel form.

Compositions comprising sheet-like ECM materials (optionally incombination with other forms of ECM materials) may be used in a varietyof applications. For example, a composition suitable for treating burnsor skin lesions may comprise two or more layers of different ECMsheet-like materials (e.g., spleen ECM and lung ECM in sheet form), forexample, wherein the sheets may be meshed or unmeshed. Silver (e.g.,elemental or ionic silver) may be added as a coating or incorporatedinto the sheets to provide antimicrobial properties.

Further, for example, the composition may comprise a vascular patch orgraft comprising one or more inner layers of lung ECM in sheet form, andone or more outer layers of spleen ECM in sheet form. For example, lungECM may comprise the innermost 1-3 layers to provide a less permeablebarrier and allow for faster endothelial cell migration across the innerportion of the patch/graft, while also utilizing the elastin in the lungECM for better compliance matching of the patch/graft to the nativeblood vessel wall. Spleen ECM may comprise the outermost 1-3 layers toprovide additional growth factors and a more porous outer layer to allowfor faster host tissue incorporation.

In some embodiments, the composition may comprise a dura mater repairpatch, comprising one or more inner layers of lung ECM in sheet form,and an outer layer of spleen ECM in particulate or gel form coated ontothe sheet. The lung ECM sheet(s) may provide a relatively smooth andless porous surface adjacent to the cerebral spinal fluid, while thespleen ECM particulate or gel may provide more growth factor content forsignaling.

In some embodiments, the composition may comprise a cornea repair patch,comprising one or more layers of lung ECM (e.g., in thin sheet form)combined with spleen ECM particulates. The lung ECM component may act asa patch while the particulate spleen ECM component may degrade morerapidly to provide a bolus of growth factor or other signalingcomponents released at the injury/repair site.

The composition may comprise a guided bone regeneration (GBR) graft orguided tissue regeneration (GTR) graft. For example, the composition maycomprise lung and spleen ECM in sheet form, the lung ECM placed adjacentto the bone to provide a less permeable barrier maintaining space forthe bone to grow, and the spleen ECM comprising outer layers to providea more porous ECM scaffold that allows for faster tissue ingrowth.

In some embodiments, the composition may comprise a hernia repair graftcomprising multiple layers of both lung and spleen ECM materials insheet form to provide sufficient strength for the specific herniarepair. Inguinal, hiatal, and ventral hernias each have differentstrength requirements due to the stresses at those particular anatomicalsites. More strength is typically required for a ventral hernia repairdevice than a hiatal hernia repair device, for example. One side of thecomposition may comprise multiple layers of lung ECM sheet material toprovide a less porous surface to minimize adhesion formation, while theopposite side may comprise multiple layers of spleen ECM sheet materialto provide a more porous interface for rapid tissue ingrowth into thegraft. The composition may comprise silver (e.g., elemental or ionicsilver) and/or one or more other antimicrobial agents to provideantimicrobial properties.

In some embodiments, the composition may have a tubular or rod-likeshape. For example, sheet-like ECM materials may be rolled or otherwisemanipulated into a tubular configuration or rod-like shape. Tubularand/or rod-like ECM compositions may be tailored for a variety ofanatomical applications.

For example, the composition may comprise a tubular nerve guide/graftincluding one or more inner layers of lung ECM sheet material and one ormore outer layers of spleen ECM sheet material. The smoother surface ofthe inner lung ECM portion may provide a conduit suitable for the nerveto grow together, for example, while the outer spleen ECM portion mayallow for incorporation of the graft into surrounding tissue. In someembodiments, the tubular ECM composition may further comprise one ormore ECM materials inside the tubular surface (e.g., an ECM particulatematerial, ECM gel, or combination ECM particulate/gel), e.g., whereinthe inside ECM material may degrade over time to release growth factorsto stimulate tissue growth, such as new nerve growth. Exemplaryapplications for tubular ECM compositions include, but are not limitedto, vascular grafts, vascular access devices, nerve guides, bile ductrepair, ureter repair, urethra repair, and fallopian tube repair.

As mentioned above, the composition may comprise ECM sheet materialrolled up into cylindrical shape, or twisted or braided into a strand orrod-like shape. In some embodiments, compressed ECM particulate,particulate/gel, or other suspension, dispersion, or fluidized ECMmaterial may be placed into a mold and compressed into a rod-like shapeor other three dimensional shape. The rod-like ECM material may includeone or more other components, such as additional bioactive components,pharmaceutical agents, other ECM materials, polymers, hydrogels,hyaluronic acid, or allograft materials. Exemplary applications forrod-like ECM compositions include, but are not limited to, tissuegrafts, tissue augmentation, and tissue reconstruction.

In some embodiments, the composition may comprise ECM materials in arod-like shape, e.g., for use as a nerve graft or other tissue graft. Anexemplary rod-like or cylindrical composition 30 is illustrated in FIGS.3A-3C. For example, the composition 30 may comprise one or more layersof ECM material rolled into a generally cylindrical shape, as shown inFIG. 3A. FIGS. 3B and 3C show cross-sections of different possibleconfigurations of the composition 30.

In some embodiments, for example, spleen ECM in sheet form may be rolledinto a cylinder, with lung ECM in sheet form forming one or more outerlayers surrounding the spleen ECM. This configuration is illustrated inFIG. 3B, showing one or more outer layers 32 of sheet-like lung ECM, andone or more inner layers 34 of reticular spleen ECM. In someembodiments, the composition 30 may comprise spleen ECM inside with lungECM outside twisted or braided into a rod-like shape as well. The moreporous portion of the spleen ECM on the interior of the composition 30may provide for rapid tissue ingrowth through the center of the graft.The less porous lung ECM on the outside may provide for rapid cellmigration across the surface of the graft while also preventing adhesionto native tissue and/or tissue ingrowth into the nerve gap beingregenerated within the interior. FIG. 3C shows a cross-section ofanother exemplary configuration of the composition 30, comprising one ormore outer layers 36 of sheet-like ECM material (e.g., sheet-like lungECM), and one or more inner layers 38 of reticular ECM materialreticular spleen ECM). In some embodiments, allograft nerve tissue maybe incorporated into the composition 30, e.g., as part of a twisted orbraided outer strand, or as the center of a rolled cylinder rod shapenerve graft.

Rod-like compositions also may be used in tissue augmentation, e.g.,during plastic surgery or reconstructive surgery. For example, thecomposition may be used in lip augmentation. Lung ECM may be used as theinterior layers of a rod-shaped composition to form a less compressiblecore of the rod, allowing it to keep its shape and bulk, while spleenECM may be used on the outside, so that the more porous spleen ECMsurface faces outwards. The porous spleen ECM may allow for rapid tissueintegration into the graft, resulting in less migration of the graft andfaster healing.

Further, for example, the composition may have a cylindrical or rod-likeshape used for nipple reconstruction. Depending on the surgicalprocedure used, the composition may be constructed with lung ECM on theinterior to provide a less porous core, and spleen ECM on the outside toprovide a more porous surface that can integrate with host tissue tokeep the graft in place and maintain a protruding shape. In someembodiments, the composition may comprise spleen ECM on the inside,e.g., for tissue integration into the center of the composition toprovide stability, and lung ECM on the outside, e.g., to provide forless host tissue invasion and better preserve the shape and volume ofthe protrusion. In some embodiments, the composition may comprise atwisted or braided strand of both lung ECM and spleen ECM.

In some embodiments, rod-like ECM material may be used as a tendon orligament graft. For example, the composition may comprise spleen ECM andlung ECM in different amount, such as a spleen:lung ratio of about70:30, 80:20, or 90:10, wherein spleen ECM comprises the inside layers,and lung ECM comprises the outer layers. As mentioned above, lung ECMgenerally has a higher elastin content and stretches more than spleenECM. A tendon or ligament graft typically requires minimal stretching toprevent the joint from becoming “loose” over time due to the tendon orligament stretching after implantation of the graft. However, duringhealing, ECM grafts often shrink slightly. An amount of stretchingtherefore may be desired, e.g., by incorporating lung ECM within theouter layers of the graft. The amount of lung ECM incorporated into thegraft may be adjusted to achieve the degree of stretching desired. Insome embodiments, the graft composition may comprise allograft tendon orligament. Incorporating ECM materials, e.g., in sheet, particulate,and/or gel forms, into the tendon or ligament graft composition mayprovide additional scaffolding and signaling components forincorporation of the tendon/ligament into the host tissue and for cellmigration and proliferation in the composite graft material.

The composition may comprise a compressible or expandable, rod-likeshape, e.g., for implantation as a medical device, e.g., for vascularaccess site repair. Typically, compression is applied to vascular accesssites to prevent bleeding and close the site. A rod-like or cylindricalcomposition comprising spleen and lung ECM (and/or other types of ECMmaterials) may be inserted into the access site, e.g., to fill and/orseal an incision in a blood vessel and stop any bleeding, while alsoproviding a scaffold and biological signaling components to heal theincision into the blood vessel. For example, the composition maycomprise a rolled cylinder with one or more inner layers of lung ECM(e.g., rolled sheets of lung ECM) to provide a less compressible ornon-compressible core, and one or more outer layers comprising moreporous spleen ECM in sheet form. The composition may be compressed intoa cylindrical shape configured to expand when wetted with blood at thesite, thus sealing the incision. Further, for example, lung and spleenECM in gel form may be dried and/or crosslinked into a cylindricalshape, wetted, compressed, and dried again to produce an expandablesponge having a generally cylindrical or rod-like shape.

In some embodiments, the composition may comprise a medical device thatincludes one or more features configured to anchor the device uponimplantation in the body, e.g., as a fistula plug. For example, highoutput fistula defects can cause fistula plugs or othermaterials/devices to dislodge from the defect. To address this problem,the device may comprise a combination of spleen and lung ECM materialsin a cylindrical or rod-shaped body portion with protrusions along thelength of the cylindrical/rod-shaped body to anchor the device againstthe walls of the fistula and prevent the device from becoming dislodged.This anchoring may also provide for close tissue approximation to allowfor tissue ingrowth into the ECM materials (e.g., ECM matrix) via thescaffolding structure and the biological signaling components.

In some embodiments, for example, the device may have an “umbrella”shape formed by expandable arms at one or both ends of the device. Wheninserted into the fistula and deployed, the “umbrella” end may open intoa disc or similar shape coupled to the cylindrical/rod-shaped bodyportion in order to seal one end of the fistula and anchor the device,e.g., to repair a defect. This configuration is illustrated in FIGS. 4Aand 4B, showing a medical device 40 configured for anchoring uponimplantation in a fistula or other passage within the body. The device40 may include a body portion 41 and at least one anchoring portion 43.For example, the body portion 41 may comprise a composition inaccordance with FIG. 3A, e.g., comprising inner and outer layers ofdifferent ECM materials, or any other combination of ECM materials asdescribed herein. The anchoring portion 43 may comprise a set ofexpandable arms 45 on opposite sides of the body portion 41. In someembodiments, the device 40 may comprise more than one anchoring portion43.

Each of the body portion 41 and the anchoring portion 43 may compriseone or more ECM materials and/or one or more non-ECM materials. Non-ECMmaterials may include, but are not limited to, biocompatible metals,metal alloys, ceramics, polymers, or combinations thereof. Suitablepolymers include, but are not limited to, polylactic acid (PLA),polyglycolide (PGA), and poly(lactic-co-glycolic acid) (PLGA). The bodyportion 41 may comprise the same or different materials as the anchoringportion 43. For example, the body portion 41 may comprise a combinationof two or more different ECM materials, and the anchoring portion 43 maycomprise at least one non-ECM material, such as a polymer or combinationof polymers. In some embodiments, the body portion 41 and the anchoringportion 43 both may comprise at least two different ECM materials. Anyof the ECM materials, combinations of ECM materials, and non-ECMmaterials (including antimicrobial agents) as discussed above may beused for the medical device 40.

When the device 40 is in a compressed configuration, as shown in FIG.4A, the arms 45 may extend substantially alongside the body portion 41to facilitate insertion into a fistula 100 or other passageway withinthe patient's body. The arms 45 may be configured to expand away fromthe body portion 41 via self-expansion once no longer restrained by thewalls of the fistula 100. Once expanded, as shown in FIG. 4B, the arms45 may anchor the device 40 within the fistula 100.

Further, for example, the composition may comprise a medical device witha cylindrical or rod-like body portion that includes flanges, “wings,”or protruding edges along at least a portion of the length of the body.FIGS. 5A and 5B illustrate an exemplary medical device 50 including abody portion 51 and one or more protrusions 55 along the length of thebody portion 51. The body portion 51 may comprise two or more differenttypes of ECM materials, such as the composition 30 of FIG. 3A, or anyother combination of ECM materials and non-ECM materials as describedherein, including the materials discussed above in connection to medicaldevice 40. The protrusions 55 may be angled in a proximal direction(i.e., away from the direction of insertion), such that the device 50may be advanced within the fistula 100 but not withdrawn, due to theprotrusions 55 catching tissue along the wall of the fistula 100. Insome embodiments, the protrusions 55 may have a pointed or barbed tip tofurther anchor the device 50 within the fistula 100.

Inhalation

In some embodiments, the composition may comprise ECM materials inparticulate form, e.g., for administration via inhalation. The particlesmay range from about 100 nm to about 5 μm in diameter, such as fromabout 1 μm to about 5 μm in diameter, or from about 100 nm to about 2.5μm in diameter. In some embodiments, for example, the composition maycomprise particles having substantially the same size, e.g., a particlediameter of about 100 nm, about 300 nm, about 500 nm, about 1 μm, about2.5 μm, or about 5 μm. In some embodiments, the composition may compriseparticles having a size distribution with an average or median diameterranging from about 300 nm to about 5 μm, e.g., about 300 nm, about 500nm, about 1 μm, about 2.5 μm, or about 5 μm. The size distribution maybe unimodal or bimodal.

The composition may comprise two or more ECM materials in particulateform. For example, the composition may comprise a first set of particlesof a first ECM material, and a second set of particles of a second ECMmaterial, wherein the first and second sets of particles may have thesame or different diameters or size distributions. In some embodiments,the composition may comprise particles of lung and spleen ECM rangingfrom about 1 μm to about 5 μm in diameter for delivery to the lungs of apatient via inhalation.

The particulate composition may be used to treat the respiratory system,to treat a respiratory disorder, and/or to deliver a pharmaceuticalagent such as a steroid for uptake via the respiratory system. In someembodiments, for example, the composition may be used to treat asthma,lung infections, or a lung injury, such as damage to the lungs frominhalation of damaging gases, smoke, or chemical or biological agents.The composition may be inhaled after exposure to toxins and harmfulsubstances to help heal damaged tissue in the lungs, and to minimizeand/or prevent lung fibrosis. In some embodiments, the composition maybe administered to mitigate the effects of diseases that cause damage tothe lungs, e.g., as part of a routine dosing regimen.

In some embodiments, the composition may serve as a delivery vehicle fora pharmaceutical or therapeutic agent. For example, the pharmaceuticalor therapeutic agent may be incorporated into the ECM material duringprocessing such that the agent is associated with the particles, such asa coating. In at least one embodiment, the composition may comprise atleast one ECM material in combination with fluticasone propionate andsalmeterol, optionally in combination with a pharmaceutical agent suchas a steroid, formulated for administration via inhalation.

Injection

The composition may be formulated for injection, e.g., in liquid or gelform. ECM particulates, gels, and/or particulate and gel combinationsmay be used as an injectable for a variety of applications, including,but not limited to arthritis (e.g., joint injections to treatarthritis), tendon/ligament partial tears, cartilage tears or damage,nerve tears or damage, myelin sheath regeneration, hair restoration, andin plastic and reconstructive surgery (e.g., as a void filler or bulkingagent). As mentioned above, ECM gels may be produced by digesting (e.g.,via digestive enzymes) or chemically processing ECM materials into gelform, which may be combined with ECM particles or other additional ECMmaterials.

In some embodiments, the composition may comprise a 50-50 ratio ofparticulate lung ECM and particulate spleen ECM suspended within an ECMgel as a carrier (e.g., a lung ECM gel, spleen ECM gel, or lung/spleenmixture ECM gel). The lung/spleen composition may be injected at one ormore sites along a nerve that has been damaged or sustained loss of themyelin sheath, such as in amyotrophic lateral sclerosis (ALS) disease ormultiple sclerosis. Without being bound by theory, it is believed thatgrowth factors and other signaling components within the ECM compositionmay signal mesenchymal stem cells to migrate to the site of injectionand induce formation of new myelin sheath or nerve tissue.

Three-Dimensional Constructs

In some embodiments, the composition may comprise ECM materials formedinto a three-dimensional construct, e.g., to fill or repair largedefects, or in patient-specific reconstruction of lost or damagedtissue. Such constructs may be useful in reconstructive surgery, whereinthere may be a need to fill a void, or to regenerate tissue in aspecific area, e.g., having a particular shape.

In some embodiments, for example, the ECM constructs may havethree-dimensional shapes obtained via specialized forms or molds, or bycutting, trimming, or otherwise manipulating larger, standardized shapessuch as blocks or rods into the desired form, e.g., to match the sizeand shape of the void or defect to be filled. The construct may vary incomposition, e.g., comprising a composite of two or more ECM materials,and may vary in porosity. The construct may comprise one or more non-ECMcomponents, including, but not limited to, cells, pharmaceutical agents,polymers, or demineralized bone.

In some embodiments, scanning, imaging techniques, and/or 3D computermodeling may be used to prepare a replica of the void/defect orotherwise obtain the dimensions of the ECM construct needed for repair.In this way, a three dimensional construct may be made according to theexact dimensions needed for a given patient. The mold may be filled withdifferent types of ECM materials and non-ECM materials to obtain apatient-specific implant.

For example, a patient with oral cancer may require surgicalintervention to remove bone and soft tissue at the site of the cancer.To repair the affected area after surgery, e.g., by implantingreplacement tissue or a tissue graft, a mold may be designed by imagingthe removal site. A combination of lung ECM and spleen ECM inparticulate and gel form may be added to the mold, e.g., to comprise thesoft tissue portion, along with demineralized bone, e.g., to comprisethe replacement jaw bone.

Additional combinations of ECM materials may include ECM sheets combinedwith one or more other ECM materials in particulate, gel, foam, and/orsponge form to create “sheet-like” constructs with varying densities andcompressibility. The constructs may be tailored for specificapplications, including, but not limited to, cartilage repair grafts andspinal disc grafts. In some embodiments, for example, the compositionmay comprise circular sheets of lung and/or spleen ECM materials withother forms of ECM materials (e.g., in particulate, gel, particulateplus gel, foam, or sponge form, or similar materials) that provide aninner circular portion that is more compressible than the outer “ring”of the lung/spleen ECM material. This configuration may simulate theannulus on the outside (e.g., the anlus fibrosus) and the nucleus on theinside (e.g., the nucleus pulposus). In some embodiments, thecomposition may be a hemostasis device comprising an outer layer of lungECM (e.g., to provide liquid impermeability), an inner layer of spleenECM (e.g., processed to maintain the unique architecture of thereticular spleen ECM structure), and a coating of lung/spleen ECMparticulate mix incorporated into the inner spleen ECM layer. The ratioof the particulate may be about 50:50 lung-spleen.

Further, the composition may be used as a bone graft material. Forexample, the composition may comprise a particulate mix of two or moreECM materials (e.g., spleen and lung ECM) in various ratios, or a mix ofan ECM material in particulate form and an ECM material in gel form(e.g., spleen ECM particulate and lung ECM gel, or lung ECM particulateand spleen ECM gel). The composition may include more or more additionalagents or materials such as, e.g., tricalcium phosphate, hydroxyapatite,bioactive glass, mineralized bone, demineralized bone, or anothermineral component. In some embodiments, the composition may include oneor more antimicrobial agents such as, e.g., ionic silver or elementalsilver, for use in bone infection healing applications.

The ECM materials may be combined or incorporated into each other and/orcombined with non-ECM materials via any suitable method, and may bephysically attached and/or chemically bonded together. For example, anadhesive such as fibrin glue, cyanoacrylate, thrombin/gelatin,polyethylene glycol (PEG), or a solder such as albumin (e.g., appliedwith laser energy or heat to weld tissues together) may be used toincorporate ECM materials into each other. Further, cross-linking agentssuch as, e.g., glutaraldehyde, dendrimers, or methylene blue, may beused to form bonds between different ECM materials. Additionally oralternatively, ECM materials may be combined in a composition viathermal energy, ultra-violet light, and/or chemical cross-linking. Forexample, sugar-based molecules such as a saccharide (e.g. glucose orgalactose) or a disaccharide (e.g. lactose or sucrose) and/orpeptide-based molecules may be used to combine different ECM materials.In some embodiments, different ECM materials may be combined into athree-dimensional construct form by exposing the materials to heat(e.g., a temperature ranging from about 50° C. to 250° C.) while theyare compressed or in close proximity to each other. The heat maygenerate bonds (crosslinks) between collagens in the different ECMmaterials.

In some embodiments, the composition may be prepared by combining an ECMmaterials in gel form with an ECM material in particulate form, e.g., toproduce a particulate wafer or cake. For example, the ECM gel may beintroduced into a mold with the ECM particulate, compressed and/orcross-linked together, and dried in a mold to form the composition in aparticulate wafer or cake. In some embodiments, the ECM gel may beintroduced into a mold without ECM particulate, and compressed and/orcross-linked to form the composition.

Compositions according to the present disclosure may be prepared bymixing different ECM materials together in particulate form. ECMparticulates may be prepared by chopping, cutting, pulverizing, milling,or grinding the ECM material with a suitable device such as a blender, ahammer mill, a knife mill, a centrifugal mill, or a roller crusher, forexample, to form particles. In some embodiments, two or more ECMmaterials in particulate form may be mixed together in a gel (e.g., anECM material in gel form, which may be the same or different type of ECMmaterial as the ECM particulates) or a liquid carrier. Examples ofsuitable carriers include, but are not limited to, hyaluronic acid,gelatin, lecithin, collagen gel, and saline.

Further, compositions according to the present disclosure may beprepared by coating one type of ECM material onto a different type ofECM material. For example, a first ECM material (e.g., an ECM core) maybe coated with a second ECM material (e.g., an ECM coating). Forexample, the composition may comprise ECM particles having a core of onetype of ECM material and a coating of a different type of ECM material.Coating the ECM core may be performed, for example, by precipitating thesecond ECM material onto the first ECM material from a solution, by aroll coater, or by a granulator. The second ECM material used as thecoating may be in a liquid or gel form. Other materials suitable coatingECM materials may include, but are not limited to, PLGA, hyaluronicacid, collagen, or a mixture thereof.

In some embodiments, the composition may comprise ECM materials in gelform, wherein the gel may change in response to a stimulus. For example,the gel may expand in vivo when injected into a target site and/or maypolymerize in vivo, and thus may stay fixed into position whenpolymerized. The stimulus may be, e.g., a change in temperature and/orpH. In some embodiments, for example, the gel may comprise two or moredifferent ECM materials and at least one temperature-responsive polymer,copolymer, or block copolymer. For example, the gel may polymerize aftera change in pH, wherein the pH may be altered by adding an acid or basesuch that the gel polymerizes in vivo at the application site. In someembodiments, the gel may be designed to polymerize at body pH afterapplication to the desired site. Different ECM materials may bedelivered to the target site concurrently in gel form, in solution, oras particulates suspended in a carrier, such that once the ECM materialsare mixed together at the target site, they polymerize and/or expand.

Other embodiments of the present disclosure will be apparent to thoseskilled in the art from consideration of the specification and practiceof the embodiments disclosed herein. While various examples providedherein illustrate specific types of ECM materials and combinationsthereof, one of ordinary skill in the art will recognize that othertypes of ECM materials from any tissue source and combinations of ECMmaterials also may be used. Further, any features of an embodimentdisclosed herein may incorporated into any other embodiment.

The following examples are intended to illustrate the present disclosurewithout, however, being limiting in nature. It is understood that thepresent disclosure encompasses additional embodiments consistent withthe foregoing description and following examples.

EXAMPLES Example 1

Samples of porcine tissues were extracted to measure growth factorcontent. Dried micronized matrix material sampled from pig stomach,spleen, and lung tissues were extracted for 72 hours at 4° C. Threedifferent buffers were used in the extractions: phosphate bufferedsaline (PBS), RIPA buffer, and 0.5 M acetic acid (AA). Each extractionincluded a 20:1 extraction volume to tissue weight ratio (6.0 mlextraction buffer for 300 mg matrix material). Extracts were analyzedfor VEGF-A, EGF, bFGF, PDGF, IGF1, and TGFβ by ELISA using theindividual ELISA instruction manual. In order to assay acetic acidextracted samples using ELISA, acetic acid extracts were neutralized byadding 219 μl of 2.5 M Tris base per 1.0 ml of acetic acid extract.Results are shown in Table 2.

TABLE 2 Growth factor concentrations (ng/ml) measured in porcinestomach, spleen, and lung tissues. Stomach Spleen Lung Extraction PBSRIPA AA PBS RIPA AA PBS RIPA AA VEGF 435.8 ± 314.7 ± 523.7 ± 411.1 ± 328± 437.2 ± 190.1 ± 157.8 ± 569.6 ± 18.6 8.7 78.9 7.3 19.4 62.4 10.9 6.1136.8 EGF 177.1 ± 96.3 ± 112.2 ± 260.4 ± 249.2 ± 206.8 ± 252.7 ± 178.1 ±174.6 ± 117.8 36.6 38.2 31.6 31.7 24.5 78.2 38.2 51.2 bFGF * * 28.7 * *50.1 * *  54.0 PDGF 303.1 * 168.8  324.5 * 318.6  303.2 * 245.3 IGF1 *3.1 20.8  3.1 11 79.6  1.7 6.4 141.7 * not detected

It is intended that the specification and examples be considered asexemplary only, with a true scope and spirit of the present disclosurebeing indicated by the following claims.

1-30. (canceled)
 31. A composition comprising: spleen extracellularmatrix in particulate form, wherein the spleen extracellular matrixcomprises native growth factors retained from spleen tissue; a secondextracellular matrix other than spleen extracellular matrix inparticulate form; and a carrier in liquid or gel form; wherein thecomposition is formulated for application onto or into a site of defector wound of a patient.
 32. The composition of claim 31, wherein thecarrier comprises a third extracellular matrix in gel form, the thirdextracellular matrix being chosen from spleen extracellular matrix, lungextracellular matrix, gall bladder extracellular matrix, bone marrowextracellular matrix, pancreatic extracellular matrix, or liverextracellular matrix.
 33. The composition of claim 31, wherein thecarrier comprises hyaluronic acid, gelatin, lecithin, collagen gel, orsaline.
 34. The composition of claim 31, wherein the spleenextracellular matrix comprises at least two native growth factorsretained from spleen tissue chosen from vascular endothelial growthfactor, platelet-derived growth factor, epidermal growth factor,fibroblast growth factor, and insulin-like growth factor.
 35. Thecomposition of claim 31, further comprising at least one of tricalciumphosphate, hydroxyapatite, bioactive glass, mineralized bone, ordemineralized bone.
 36. The composition of claim 31, further comprisingat least one of an antimicrobial agent or a pharmaceutical agent. 37.The composition of claim 31, formulated as a coating of an implantablemedical device or formulated for injection into the site of defect orwound of the patient.
 38. A composition comprising: spleen extracellularmatrix in particulate form, wherein the spleen extracellular matrixcomprises at least two native growth factors retained from spleen tissuechosen from vascular endothelial growth factor, platelet-derived growthfactor, epidermal growth factor, fibroblast growth factor, andinsulin-like growth factor; lung extracellular matrix in particulateform; and a carrier comprising a third extracellular matrix in gel form;wherein the composition is formulated for injection into a site ofdefect or wound of a patient.
 39. The composition of claim 38, whereinthe particles of the spleen extracellular matrix range from about 100 nmto about 2000 μm in diameter.
 40. The composition of claim 38, whereinthe particles of the spleen extracellular matrix range from about 50 μmto about 100 μm in diameter.
 41. The composition of claim 38, whereinthe particles of the spleen extracellular matrix or the particles of thelung extracellular matrix have a bimodal size distribution.
 42. A methodfor treating a patient, comprising: delivering a composition to a siteof defect or wound of the patient, wherein the composition comprises:spleen extracellular matrix in particulate form, wherein the spleenextracellular matrix retains native growth factors of spleen tissue; asecond extracellular matrix other than spleen extracellular matrix inparticulate form; and a carrier in liquid or gel form.
 43. The method ofclaim 42, wherein the carrier comprises a third extracellular matrix ingel form, the third extracellular matrix being chosen from spleenextracellular matrix, lung extracellular matrix, gall bladderextracellular matrix, bone marrow extracellular matrix, pancreaticextracellular matrix, or liver extracellular matrix.
 44. The method ofclaim 42, wherein the particles of the spleen extracellular matrix rangefrom about 50 μm to about 100 μm in diameter, and wherein the spleenextracellular matrix comprises at least two native growth factorsretained from spleen tissue chosen from vascular endothelial growthfactor, platelet-derived growth factor, epidermal growth factor,fibroblast growth factor, and insulin-like growth factor.
 45. The methodof claim 42, wherein the composition further comprises at least one oftricalcium phosphate, hydroxyapatite, bioactive glass, mineralized bone,or demineralized bone.
 46. The method of claim 42, wherein thecomposition is injected into a joint of the patient.
 47. The method ofclaim 42, wherein the composition is injected into cartilage, a tendon,or a ligament of the patient.
 48. The method of claim 42, wherein thecomposition is delivered to one or more sites along a nerve that hasbeen damaged or sustained loss of a myelin sheath of the nerve.
 49. Themethod of claim 42, wherein the composition is injected into myocardialtissue of the patient.
 50. The method of claim 42, wherein thecomposition is delivered to an eye of the patient.